SRM Assay to Indicate Cancer Therapy

ABSTRACT

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins that are particularly advantageous for quantifying the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins directly in biological samples that have been fixed in formalin by the methods of Selected Reaction Monitoring (SRM) mass spectrometry, or as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from the biological sample using the Liquid Tissue™ reagents and protocol and the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins are quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry, by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an ALK, Ros, Ron, Ret, TS, and/or FGFR1 fragment peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application is a divisional of application Ser. No. 14/855,154, nowU.S. Pat. No. 9,840,728, which is a continuation of InternationalApplication No. PCT/US14/31138, filed Mar. 18, 2014, which claims thebenefit of U.S. Provisional Application No. 61/791,833, filed Mar. 15,2013, each entitled “SRM Assay to Indicate Cancer Therapy.” The contentsof each of which are hereby incorporated by reference in theirentireties. This application also contains a sequence listing submittedelectronically via EFS-web, which serves as both the paper copy and thecomputer readable form (CRF) and consists of a file entitled“3900_0013DIV_SEQ_LISTING”, which was created on Dec. 8, 2017, which is6,345 bytes in size, and which is also incorporated by reference in itsentirety.

INTRODUCTION

Lung cancer is the most prevalent cancer (>200,000 new US cases/year)and has a low five-year survival rate (˜15%). Therapy for lung cancer istransitioning from use of a limited selection of therapies, consistingof radiation, folate metabolism, platinum-based drugs, and/ortaxol-based drugs, to more targeted treatments that require histologicalcharacterization of the tumor and/or the presence or absence of keybiomarker or therapeutic target proteins. A full 80% of all lung cancersare of the non-small cell lung cancer (NSCLC) type and this general typecan be broken down into 4 different subtypes based on histologicalanalysis: adenocarcinoma, squamous cell carcinoma, bronchioalveolarcarcinoma, and Large-cell undifferentiated carcinoma. The remaining 20%of lung cancers are known as small cell lung cancer (SCLC).Recently-utilized targeted cancer therapies have shown exciting successin treating both NSCLC and SCLC patients. A targeted approach to cancertherapy is most advantageous when growth of the cancer is driven by aspecific target protein, or proteins, and where the target protein, orproteins, that are driving the cancer are specifically attacked bytherapeutic agents designed and manufactured to inhibit the targetprotein, or proteins, and hence inhibit growth of the cancer. Peptidesand peptide sequences are provided for use in an SRM/MRM assay which canbe used to quantitatively determine which protein targets are expressed,or over-expressed, directly in tumor tissue derived from lung cancerpatients. This permits improved treatment decisions for targeted lungcancer therapy. Also provided is an SRM/MRM assay which can be used toquantitatively determine which protein targets are expressed, orover-expressed, directly in tumor tissue derived from patients withcancers other than lung cancer for improving targeted treatmentdecisions for any other cancer that is not lung cancer.

SUMMARY

Specific peptides derived from subsequences of the following proteinsare provided, ALK, Ros, Ron, Ret, TS, and FGFR1. ALK is also known asALK tyrosine kinase receptor and CD246 and is referred to herein as ALK.Ros is also known as c-Ros receptor tyrosine kinase and is referred toherein as Ros. Ron is also known as Macrophage-stimulating proteinreceptor, CD136, and p185-Ron, and is referred to herein as Ron. Ret isalso known as Proto-oncogene tyrosine-protein kinase receptor Ret andProto-oncogene c-Ret, and is referred to herein as Ret. TS is also knownas thymidylate synthase, and is referred to herein as TS. FGFR1 is alsoknown as fibroblast growth factor receptor 1 and CD331, and is referredto herein as FGFR1.

The peptide sequence and fragmentation/transition ions for each peptidederived from proteins are potentially useful in a massspectrometry-based Selected Reaction Monitoring (SRM) assay(s), whichcan also be referred to as a Multiple Reaction Monitoring (MRM)assay(s), hereinafter referred to as SRM/MRM assay(s). The use ofpeptides for SRM/MRM analysis of ALK, Ros, Ron, Ret, TS, and FGFR1proteins and isoforms of those proteins is described.

One or more, two or more, three or more, four or more, or five or sixSRM/MRM assay(s) can be used to detect the presence and measure relativeor absolute quantitative levels of one or more of the specific peptidesfrom the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins, and therebyprovide a means of measuring the total amount of each of those proteinsin a given protein preparation obtained from a biological sample by massspectrometry. All, or a portion of all of the available peptides fromthose proteins can also be analyzed simultaneously in a single SRM/MRMassay or can be analyzed in any combination of individual SRM/MRMassays. Each of the peptides provides a potential means of measuring thetotal amount of each of the corresponding proteins in a given proteinpreparation obtained from a biological sample by mass spectrometry.

The SRM/MRM assay(s) described herein can measure these peptidesdirectly in complex protein lysate samples prepared from cells procuredfrom patient tissue samples, such as formalin fixed cancer patienttissue (e.g., biopsies). Methods of preparing protein samples fromformalin fixed tissue are described in U.S. Pat. No. 7,473,532, thecontents of which are hereby incorporated by reference in theirentirety. The methods described in that patent may conveniently becarried out using Liquid Tissue reagents and protocol available fromExpression Pathology Inc. (Rockville, Md.).

Formaldehyde/formalin fixation of tissues surgically removed from cancerpatients is the accepted convention in pathology practice. As a result,formaldehyde/formalin fixed paraffin embedded tissue is the most widelyavailable form of tissues from those patients. Formaldehyde/formalinfixation typically employs aqueous solutions of formaldehyde referred toas formalin. “100%” formalin consists of a saturated solution offormaldehyde (about 40% formaldehyde by volume or 37% by mass) in water,with a small amount of stabilizer, usually methanol, to limit oxidationand degree of polymerization. The most common way in which tissue ispreserved is to soak whole tissue for extended periods of time (8 hoursto 48 hours) in aqueous formaldehyde, commonly termed 10% neutralbuffered formalin, followed by embedding the fixed whole tissue inparaffin wax for long term storage at room temperature. Thus molecularanalytical methods to analyze formalin fixed cancer tissue will be themost accepted and heavily utilized methods for analysis of cancerpatient tissue.

Results from the SRM/MRM assay(s) can be used to correlate accurate andprecise quantitative levels of any or all of these proteins, in additionto accurate and precise quantitative levels of potential isoforms ofthese proteins, within specific tissue samples (e.g., cancer tissuesample) of a patient or subject from whom the tissue (biological sample)was collected and preserved. This not only provides diagnosticinformation about the cancer, but also permits a physician or othermedical professional to determine appropriate therapy for the patient orsubject. Such an assay that provides diagnostically and therapeuticallyimportant information about levels of protein expression in a diseasedtissue or in another patient/subject sample is termed a companiondiagnostic assay. For example, such an assay can be designed to diagnosethe stage, degree, or histology of a cancer and determine a therapeuticagent that will be most effective in stopping the cancer cells fromgrowing, leading to the determination to which therapeutic agent that apatient or subject will most likely respond.

More specifically, detection and/or quantitation of one or more, two ormore, three or more, four or more, five or more of the ALK, Ros, Ron,Ret, TS, and/or FGFR1 proteins, in cancer cells from a patient isindicative of cancer growth, most notably lung cancer, and identifyproteins that can be targeted by targeted treatment regimens.

DETAILED DESCRIPTION

The assays described herein quantify or measure relative or absolutelevels of specific unmodified peptides from proteins including ALK, Ros,Ron, Ret, TS, and/or FGFR1 proteins and also can measure relative orabsolute levels of specific modified peptides from those proteins.Examples of modifications include phosphorylated amino acid residues andglycosylated amino acid residues that are present on the peptides.

Relative quantitative levels of proteins and potential isoforms, can bedetermined by the SRM/MRM methodology, for example by comparing SRM/MRMsignature peak areas (e.g., signature peak area or integrated fragmention intensity). Relative levels of individual ALK, Ros, Ron, Ret, TS,and/or FGFR1 peptides can be determined in different samples (e.g., acontrol sample and a sample prepared from a patient's or subject'stissue). Alternatively, where each peptide has its own specific SRM/MRMsignature peak, it is possible to compare multiple SRM/MRM signaturepeak areas for one or more of ALK, Ros, Ron, Ret, TS, and/or FGFR1signature peptides. By comparing peak areas it is possible to determinethe relative ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein and potentialprotein isoform content in one biological sample or in one or moreadditional or different biological samples. In this way, the relativeamount of a particular peptide, or peptides, from the those proteins,and therefore the relative amount of the ALK, Ros, Ron, Ret, TS, and/orFGFR1 proteins, and their potential isoforms, can be determined, acrossmultiple (e.g., two, three, four, five, or more) biological samplesunder the same experimental conditions can be determined. In addition,relative quantitation can be determined for a given peptide, orpeptides, from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein within asingle sample by comparing the signature peak area for that peptide bySRM/MRM methodology to the signature peak area for another and differentpeptide, or peptides, from a different protein, or proteins, within thesame protein preparation from the biological sample.

Using such methodologies the amount of a particular peptide from theALK, Ros, Ron, Ret, TS, and/or FGFR1 protein, and therefore the amountof each of the corresponding proteins and their potential isoforms canbe determined relative one to another within the same sample or indifferent samples. Since relative quantitation of an individual peptide,or peptides, may be conducted relative to the amount of another peptide,or peptides, within or between samples, it is possible to determine therelative amounts of the peptides present (e.g., by determining the peakarea relative one to another), regardless of the absolute weight tovolume or weight to weight amounts of the proteins in the biologicalsample. Thus, the amounts of ALK, Ros, Ron, Ret, TS, and/or FGFR1peptide in the protein preparation from the biological sample may beused to determine the amounts of those proteins in and among varioussamples. Relative quantitative data about individual signature peakareas between different samples are generally normalized to the amountof protein analyzed per sample (e.g., the total protein concentration ofa sample and the volume analyzed are used to normalize samples).Relative quantitation can be performed across many peptides frommultiple proteins and the ALK, Ros, Ron, Ret, TS, and/or FGFR1protein(s) simultaneously in a single sample and/or across many samplesto gain further insight into relative protein amounts, and/or onepeptide/protein with respect to other peptides/proteins.

Absolute quantitative levels of the ALK, Ros, Ron, Ret, TS, and/or FGFR1proteins are determined by, for example, the SRM/MRM methodology wherebythe SRM/MRM signature peak area of an individual peptide from the ALK,Ros, Ron, Ret, TS, and/or FGFR1 proteins in one biological sample iscompared to the SRM/MRM signature peak area of a known amount of one ormore internal standards “spiked” in the sample in known amounts (e.g.,isotope labeled standards). In one embodiment, the internal standard isa synthetic version of the same exact ALK, Ros, Ron, Ret, TS, and/orFGFR1 peptide that contains one or more amino acid residues labeled withone or more heavy isotopes. Such isotope labeled internal standards aresynthesized so mass spectrometry analysis generates a predictable andconsistent SRM/MRM signature peak that is different and distinct fromthe native ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptide signature peakand which can be used as a comparator peak. Thus, when the internalstandard is spiked in known amounts into a protein or peptidepreparation from a biological sample in known amounts and analyzed bymass spectrometry, the SRM/MRM signature peak area of the native peptidecan be compared to the SRM/MRM signature peak area of the internalstandard peptide. The numerical comparison permits a calculation ofeither the absolute molarity and/or absolute weight of the nativepeptide present in the original protein preparation from the biologicalsample, from which the concentration or weight of the correspondingprotein may be determined. Absolute quantitative data for fragmentpeptides are typically displayed according to the amount of proteinanalyzed per sample. Absolute quantitation can be performed across manypeptides, which permits a quantitative determination of multipleproteins (e.g., two, three, four, five, etc.) simultaneously in a singlesample and/or across multiple samples to gain insight into absoluteprotein amounts in individual biological samples and/or in entirecohorts of individual samples. In one embodiment, the quantitation ofproteins may be conducted using peptide standards as described by Gygiet al in U.S. Pat. No. 7,501,286.

Unless otherwise specified, as used herein the terms quantify,quantifying, measure or measuring mean to determine relative or absolutelevels of an analyte, such as a protein, polypeptide, peptide, astandard (e.g., an internal standard).

Assay methods described herein can be used as an aid for determining thestage of the cancer when employing, for example, patient-derived orsubject-derived tissue, such as formalin fixed tissue. The SRM/MRMassays described herein may also be used as an aid in determining whichtherapeutic agent would be most advantageous for use in treating thatpatient or subject.

To examine the levels of the proteins associated with lung cancerdescribed herein, analysis can be conducted on cancerous tissue ortissue that is suspected of being cancerous that is removed from apatient or subject, either through surgical removal of partial or entiretumors, or through biopsy procedures conducted to determine the presenceor absence of suspected disease. Samples of the tissues are analyzed todetermine whether or not one or more of ALK, Ros, Ron, Ret, TS, and/orFGFR1 protein(s), and which forms of those proteins, are present in apatient's or subject's tissue. Moreover, the expression level of one ormore of those proteins can be determined and compared to a “normal” orreference level found in healthy tissue. Normal or reference levels ofproteins found in healthy tissue may be derived from, for example, therelevant tissues of one or more individuals that do not have cancer.Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues (e.g., portionsof the same organ) not affected by the cancer.

Levels or amounts of proteins or peptides can be defined as the quantityexpressed in moles, mass or weight of a protein or peptide determined bythe SRM/MRM assay. The level or amount may be normalized to the totallevel or amount of protein or another component in the lysate analyzed(e.g., expressed in micromoles/microgram of protein ormicrograms/microgram of protein) or even normalized to the amount of DNAon a per weight basis (e.g., micromoles or micrograms/microgram of DNA).In addition, the level or amount of a protein or peptide may bedetermined on a volume basis, expressed, for example, in micromolar ornanograms/microliter. The level or amount of protein or peptide asdetermined by the SRM/MRM assay can also be normalized to the number ofcells analyzed.

Information regarding ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins, andisoforms of these proteins, can be used to aid in determininghistological stage or grade of a cancer by correlating or comparing thelevel of the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins, and theirisoforms, or fragment peptides with the levels observed in normaltissues. Once the histological stage and/or grade, and/or ALK, Ros, Ron,Ret, TS, and/or FGFR1 protein-expression characteristics of the cancerhas been determined, that information can be matched to a list oftherapeutic agents (chemical and biological) developed to specificallytreat cancer tissue that is characterized by, for example, abnormalexpression of the protein or protein(s) (e.g., ALK, Ros, Ron, Ret, TS,and/or FGFR1) that were assayed. Matching information from an ALK, Ros,Ron, Ret, TS, and/or FGFR1 protein assay from a specific individual to alist of therapeutic agents that specifically targets cells/tissueexpressing the ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein(s)represents a personalized medicine approach to treating cancer includinglung cancer. The assay methods described herein form the foundation of apersonalized medicine approach by using analysis of proteins from thepatient's or subject's own tissue as a source for diagnostic andtreatment decisions.

Peptide Generation

In principle, any predicted peptide derived from the ALK, Ros, Ron, Ret,TS, and/or FGFR1 protein, prepared by any proteolytic process of knownspecificity may be used as a surrogate reporter to determine theabundance of ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins. In oneembodiment samples are digested with a protease or proteases of knownspecificity (e.g. one or more of trypsin and/or Endoproteinase Lys-C).One or more peptides resulting from the proteolytic treatment can beused as a surrogate reporter to determine the abundance of one or moreof ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins in a suitable assaysuch as a mass spectrometry-based SRM/MRM assay. Similarly, anypredicted peptide sequence containing an amino acid residue at a sitethat is known to be modified in the ALK, Ros, Ron, Ret, TS, and/or FGFR1proteins may also be used to assay the extent of modification of ALK,Ros, Ron, Ret, TS, and/or FGFR1 proteins in a sample.

ALK, Ros, Ron, Ret, TS, and/or FGFR1 fragment peptides may be generatedby a variety of means including by the use of the Liquid Tissue™protocol provided in U.S. Pat. No. 7,473,532. The Liquid Tissue™protocol and reagents are capable of producing peptide samples suitablefor mass spectroscopic analysis from formalin fixed paraffin embeddedtissue by proteolytic digestion of the proteins in the tissue/biologicalsample. In the Liquid Tissue™ protocol the tissue/biological ismaintained at elevated temperatures in a buffer for an extended periodof time (e.g., from about 80° C. to about 100° C. for a period of timefrom about 10 minutes to about 4 hours) to reverse or release proteincross-linking. The buffer employed is a neutral buffer, (e.g., aTris-based buffer, or a buffer containing a detergent) andadvantageously is a buffer that does not interfere with massspectrometric analysis. Next, the tissue/biological sample is treatedwith one or more proteases including, but not limited to, trypsin,chymotrypsin, pepsin, and Endoproteinase Lys-C for a time sufficient todisrupt the tissue and cellular structure of said biological sample andto liquefy said sample (e.g., a period of time from about 30 minutes toabout 24 hours at a temperature from about 37° C. to about 65° C.). Theresult of the heating and proteolysis is a liquid, soluble, dilutablebiomolecule lysate. In one embodiment two or more proteases selectedfrom trypsin, chymotrypsin, pepsin, and Endoproteinase Lys-C areemployed in the proteolytic treatment of the biological sample.

Peptide Separation and Assay

Once lysates are prepared, peptides in the samples may be subject to avariety of techniques that facilitate their analysis and measurement(quantification). Where analysis is conducted by mass spectrometry, oneor more chromatographic methods may be employed in order to facilitatethe analysis.

In one embodiment the peptides are separated by liquid chromatography(LC) prior to analysis by a mass spectrometer instrument. For example,peptides can be separated on a nanoAcquityLC system (Waters, Milford,Mass.) or EASY-nLC II (Thermo Scientific, San Jose, Calif.) with aPicoFrit (100 μm ID/10 μm tip ID, New Objective) column self-packed to abed length of 12 cm with Jupiter Proteo 90 Å C12, 4 μm resin(Phenomenex, Torrance, Calif.). Peptides can be eluted over a 12 minchromatography gradient from 1% to 50% acetonitrile, containing 0.1%formic acid and at a flow rate of 800 nL/min. Once separated by liquidchromatography, the eluted peptides are directed into a massspectrometer for analysis. In one embodiment, mass spectrometer isequipped with a nanospray source.

In another embodiment, the peptides may be separated by an affinitytechnique, such as for example immunologically-based purification (e.g.,immunoaffinity chromatography), chromatography on ion selective mediaor, if the peptides are modified, by separation using appropriate mediasuch as lectins for separation of carbohydrate modified peptides. Instill another embodiment, the SISCAPA method, which employsimmunological separation of peptides prior to mass spectrometricanalysis, is employed. The SISCAPA technique is described, for example,in U.S. Pat. No. 7,632,686. In still other embodiments, lectin affinitymethods (e.g., affinity purification and/or chromatography may be usedto separate peptides from a lysate prior to analysis by massspectrometry. Methods for separation of groups of peptides, includinglectin-based methods, are described, for example, in Geng et al., J.Chromatography B, 752:293-306 (2001). Immunoaffinity chromatographytechniques, lectin affinity techniques and other forms of affinityseparation and/or chromatography (e.g., reverse phase, size basedseparation, ion exchange) may be used in any suitable combination tofacilitate the analysis of peptides by mass spectrometry.

Surprisingly, it was found that many potential peptide sequences fromthe ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins are unsuitable orineffective for use in mass spectrometry-based SRM/MRM assays forreasons that are not evident. In particular it was found that manytryptic peptides from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteinscould not be detected efficiently or at all in a Liquid Tissue™ lysatefrom formalin fixed, paraffin embedded tissue. As it was not possible topredict the most suitable peptides for MRM/SRM assay, it was necessaryto experimentally identify modified and unmodified peptides in actualLiquid Tissue™ lysates to develop a reliable and accurate SRM/MRM assayfor the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins. While not wishingto be bound by any theory, it is believed that some peptides might, forexample, be difficult to detect by mass spectrometry as they do notionize well or produce fragments distinct from other proteins, peptidesmay also fail to resolve well in separation (e.g., liquidchromatography), or adhere to glass or plastic ware. Accordingly, thosepeptides from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins that canbe detected in a Liquid Tissue™ lysate (e.g., the peptides in Tables 1and 2) prepared from a formalin fixed tissue sample are the peptides forwhich SRM/MRM assays can be employed in an ALK, Ros, Ron, Ret, TS,and/or FGFR1 proteins SRM/MRM assay. In one embodiment the proteaseemployed in the simultaneous preparation of fragments of the ALK, Ros,Ron, Ret, TS, and/or FGFR1 proteins in a single sample will be trypsin.

ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptides found in variousembodiments described herein (e.g., Tables 1 and/or 2) were derived fromthe ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by trypsin digestionof all the proteins within a complex Liquid Tissue™ lysate prepared fromcells procured from formalin fixed cancer tissue. Unless notedotherwise, in each instance the protease was trypsin. The Liquid Tissue™lysate was then analyzed by mass spectrometry to determine thosepeptides derived from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteinsthat are detected and analyzed by mass spectrometry. Identification of aspecific preferred subset of peptides for mass spectrometric analysis isbased on: 1) experimental determination of which peptide or peptidesfrom a protein ionize in mass spectrometry analyses of Liquid Tissue™lysates, and 2) the ability of the peptide to survive the protocol andexperimental conditions used in preparing a Liquid Tissue™ lysate. Thislatter property extends not only to the amino acid sequence of thepeptide but also to the ability of a modified amino acid residue withina peptide to survive in modified form during the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissue™reagents and protocol that entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue™ buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, such astrypsin, although other proteases can be used. Each protein lysate isturned into a collection of peptides by digestion of intact polypeptideswith the protease. Each Liquid Tissue™ lysate was analyzed (e.g., by iontrap mass spectrometry) to perform multiple global proteomic surveys ofthe peptides where the data was presented as identification of as manypeptides as could be identified by mass spectrometry from all cellularproteins present in each protein lysate. An ion trap mass spectrometeror another form of a mass spectrometer that is capable of performingglobal profiling, for identification of as many peptides as possiblefrom a single complex protein/peptide lysate is typically employed foranalysis. Although SRM/MRM assay can be developed and performed on anytype of mass spectrometer, including a MALDI, ion trap, or triplequadrupole, the most advantageous instrument platform for SRM/MRM assayis often considered to be a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single MSanalysis of a single lysate under the conditions employed, then thatlist of peptides was collated and used to determine the proteins thatwere detected in that lysate. That process was repeated for multipleLiquid Tissue™ lysates, and the very large list of peptides was collatedinto a single dataset. That type of dataset can be considered torepresent the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue™ lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the ALK, Ros, Ron,Ret, TS, and/or FGFR1 proteins.

In one embodiment, the ALK, Ros, Ron, Ret, TS, and/or FGFR1 trypticpeptides identified as useful in the determination of absolute orrelative amounts of: ALK (e.g., NCBI Accession No.: Q9UM73, SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5), Ros (e.g., NCBIAccession No.: P08922, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; Ron(e.g., NCBI Accession No.: Q04912, SEQ ID NO: 9, SEQ ID NO:10, SEQ IDNO:11. SEQ ID NO:12, and SEQ ID NO:13); Ret (e.g., NCBI Accession No.:P07949, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26); TS (e.g., NCBIAccession No.: P04818, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQID NO:35), and/or FGFR1 (e.g., NCBI Accession No.: P11362, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39), include one or more, twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, or ten or more or all of thepeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16. SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, each ofwhich are listed in Table 1. Each of those peptides was detected by massspectrometry in Liquid Tissue™ lysates prepared from formalin fixed,paraffin embedded tissue. Thus, each of the peptides in Table 1, or anycombination of those peptides (e.g., one or more, two or more, three ormore, four or more, five or more, six or more, or seven or more, eightor more, nine or more, or ten or more of those peptides recited inTable 1) are candidates for use in quantitative SRM/MRM assay for theALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins including directly informalin fixed patient or subject tissue.

TABLE 1 SEQ ID Protein Peptide Sequence SEQ ID NO: 1 ALK SLAVDFVVPSLFRSEQ ID NO: 2 ALK DSFPFLSHR SEQ ID NO: 3 ALK SLSAVDFFALK SEQ ID NO: 4 ALKSNQEVLEFVTSGGR SEQ ID NO: 5 ALK DPEGVPPLLVSQQAK SEQ ID NO: 6 ROSIQDQLQLFR SEQ ID NO: 7 ROS GEGLLPVR SEQ ID NO: 8 ROS EGVTVLINEDKSEQ ID NO: 9 RON ILQVELVR SEQ ID NO: 10 RON LHVLGPDLK SEQ ID NO: 11 RONVDGTSVLR SEQ ID NO: 12 RON DLISFGLQVAR SEQ ID NO: 13 RON DLDSALLAEVKSEQ ID NO: 14 RET TLGEGEFGK SEQ ID NO: 15 RET NILVAEGR SEQ ID NO: 16 RETALPSTWIENK SEQ ID NO: 17 RET ISDFGLSR SEQ ID NO: 18 RET DVYEEDSYVKSEQ ID NO: 19 RET VGPGYLGSGGSR SEQ ID NO: 20 RET AGYTTVAVK SEQ ID NO: 21RET DLLSEFNVLK SEQ ID NO: 22 RET ATAFHLK SEQ ID NO: 23 RET NLVLGKSEQ ID NO: 24 RET ILEDPK SEQ ID NO: 25 RET ADGTNTGFPR SEQ ID NO: 26 RETQVNHPHVIK SEQ ID NO: 27 TS GSTNAK SEQ ID NO: 28 TS VIDTIK SEQ ID NO: 29TS TNPDDR SEQ ID NO: 30 TS HFGAEYR SEQ ID NO: 31 TS DEFPLLTTKSEQ ID NO: 32 TS DFLDSLGFSTR SEQ ID NO: 33 TS GVLEELLWFIK SEQ ID NO: 34TS EEGDLGPVYGFQWR SEQ ID NO: 35 TS AEDFQIEGYNPHPTIK SEQ ID NO: 36 FGFR1DDVQSINWLR SEQ ID NO: 37 FGFR1 DGVQLAESNR SEQ ID NO: 38 FGFR1 LHAVPAAKSEQ ID NO: 39 FGFR1 HPAQLANGGLK

The ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptides listed in Table 1include those detected from multiple Liquid Tissue™ lysates of multipledifferent formalin fixed tissues of different human organs includingprostate, colon, and breast. Each of those peptides is considered usefulfor quantitative SRM/MRM assay of the ALK, Ros, Ron, Ret, TS, and/orFGFR1 proteins in formalin fixed tissue. Further data analysis of theseexperiments indicated no preference is observed for any specificpeptides from any specific organ site. Thus, each of these peptides isbelieved to be suitable for conducting SRM/MRM assays of the ALK, Ros,Ron, Ret, TS, and/or FGFR1 proteins on a Liquid Tissue™ lysate from anyformalin fixed tissue originating from any biological sample or from anyorgan site in the body.

In another embodiment, an SRM/MRM assay employs one or two peptides foreach of TS and FGFR1 (e.g., from the peptides listed in Table 1). Inanother embodiment an SRM/MRM assay employs one or two peptides for eachof ALK, Ros, Ron, and/or Ret (e.g., from the peptides listed in Table1).

In other embodiments one or both of the ALK and Ros proteins are assayedand one, two three or four of the Ron, Ret, TS, and/or FGFR1 protein areassayed using SRM/MRM assay(s).

In one example of such an embodiment, at least one peptide or at leasttwo peptides for one or both of the Ron and Ret protein are assayed bySRM/MRM assay (e.g., the Ron and Ret peptides listed in Table 1); and atleast one peptide or at least two peptides for any one, two, three orfour of ALK, Ros, TS, and/or FGFR1 are assayed (e.g., the peptideslisted in Table 1). In another example of such an embodiment: at leastone or at least two peptides for one or both of the ALK and Ros proteinare assayed by SRM/MRM assay (e.g., peptides listed in Table 1); and atleast one or at least two peptides for any of Ron, Ret, TS, and/or FGFR1are assayed (e.g., the peptides listed in Table 1). Compositionscomprising peptides that are isotopically labeled, but otherwiseidentical to one or more of the peptides set forth in any of theseembodiments are provided for herein and their preparation and use,particularly for use as mass spectrometry standards, is described below.

In one embodiment one or more peptides in Table 1, or any combination ofthose peptides (e.g., one or more, two or more, three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, or all eleven) is assayed by a method that does not rely upon massspectroscopy, including, but not limited to, immunological methods(e.g., Western blotting or ELISA). In one embodiment, the assays areconducted using formalin fixed tissue. Regardless of how informationdirected to the amount of the peptide(s) (absolute or relative) isobtained, the information may be employed in any of the methodsdescribed herein, including indicating (diagnosing) the presence ofcancer in a patient or subject, determining the stage/grade/status ofthe cancer, providing a prognosis, or determining the therapeutic ortreatment regimen for a patient or subject.

In other embodiments one or both of the ALK and Ros proteins are assayedand one, two three or four of the Ron, Ret, TS, and FGFR1 proteins areassayed by a method that does not rely upon mass spectroscopy,including, but not limited to, immunological methods (e.g., Westernblotting or ELISA). In one example of such an embodiment: at least oneor at least two peptides for one or both of the ALK and Ros proteins areassayed (e.g., the ALK and ROS peptides listed in Table 1); and at leastone or at least two peptides for any one, two, three or four of Ron,Ret, TS, and FGFR1 proteins are assayed (e.g., the peptides listed inTable 1). In another example of such an embodiment: at least one or atleast two peptides for one or both of the ALK and Ron proteins are(e.g., the ALK and Ron peptides listed in Table 1); and at least one orat least two peptides for any of Ros, Ret, TS, and FGFR1 proteins areassayed (e.g., the peptides listed in Table 1).

An important consideration when conducting an SRM/MRM assay is the typeof instrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,presently the most advantageous instrument platform for SRM/MRM assay isoften considered to be a triple quadrupole instrument platform. Thattype of a mass spectrometer may be considered to be the most suitableinstrument for analyzing a single isolated target peptide within a verycomplex protein lysate that may consist of hundreds of thousands tomillions of individual peptides from all the proteins contained within acell.

In order to most efficiently implement a SRM/MRM assay for each peptidederived from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins it isdesirable to utilize information in addition to the peptide sequence inthe analysis. That additional information may be used in directing andinstructing the mass spectrometer (e.g. a triple quadrupole massspectrometer) to perform the correct and focused analysis of specifictargeted peptide(s) such that the assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptides, may include one,two, three, four, or more of the mono isotopic mass of each peptide, itsprecursor charge state, the precursor m/z value, the m/z transitionions, and the ion type of each transition ion. Additional peptideinformation that may be used to develop an SRM/MRM assay for the ALK,Ros, Ron, Ret, TS, and/or FGFR1 proteins is shown in Table 2 for 12 ALK,Ros, Ron, Ret, TS, and/or FGFR1 peptides from the list in Table 1. Thisadditional information described for the peptides as shown in Table 2may be prepared, obtained, and applied to the analysis of any otherpeptides from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins,including those produced by the action of other proteases orcombinations of proteases (e.g., trypsin and/or Lys C).

TABLE 2 Mono Precursor isotopic Charge Precursor Transition IonPeptide Sequence mass State m/z m/z Type ALK SEQ ID 1 SLAVDFVVPSLFR1448.8027 2 725.409  619.356 y5 2 725.409  718.424 y6 2 725.409  817.493y7 2 725.409  964.561 y8 2 725.409 1079.588 Y9 2 DSFPFLSHR 1104.5352 2553.275  512.293 y4 2 553.275  659.362 y5 2 553.275  756.415 y6 4SNQEVLEFVTSGGR 1521.7423 2 761.878  723.378 y7 2 761.878  852.421 y8 2761.878  965.505 y9 2 761.878 1064.573 y10 Ros SEQ ID 6 IQDQLQLFR1159.6349 2 580.825 435.271 y3 2 580.825 563.33 y4 2 580.825 676.414 y52 580.825 804.472 y6 2 580.825 919.499 y7 7 GEGLLPVR  839.4865 2 420.75371.24 y3 2 420.75 484.324 y4 2 420.75 654.429 y6 Ron SEQ ID 9 ILQVELVR968.6018 2 485.308 516.314 y4 2 485.308 615.382 y5 2 485.308 743.441 y62 485.308 856.525 y7 10 LHVLGPDLK 990.5862 2 496.3 529.298 y5 2 496.3642.382 y6 2 496.3 741.45 y7 Ret SEQ ID 16 ALPSTWIENK 936.4552 2 579.811390.198 y3 2 579.811 503.282 y4 2 579.811 689.361 y5 2 579.811 790.409y6 2 579.811 877.441 y7 TS SEQ ID 31 DEFPLLTTK 1062.5597 2 532.287462.292 y4 2 532.287 575.376 y5 2 532.287 672.429 y6 2 532.287 819.497y7 35 AEDFQIEGYNPHPTI 1857.8897 3 620.304 458.297 y4 3 620.304 578.293y10 3 620.304 634.835 y11 3 620.304 692.408 y6 3 620.304 829.912 y14FGFR1 SEQ ID 36 DDVQSINWLR 1244.6149 2 623.315 588.325 y4 2 623.315701.409 y5 2 623.315 788.441 y6 2 623.315 916.499 y7 38 LHAVPAAK 805.481 2 403.748 386.239 y4 2 403.748 485.308 y5 2 403.748 556.345 y6

In some embodiments, the peptides suitable for assays of ALK, Ros, Ron,Ret, TS, and/or FGFR1 proteins (e.g., the peptides set forth in Table 1and shown as SEQ ID, Nos. 1-39) may contain additional proteolytic sitesinternal to the peptide sequences that if cleaved would producesub-peptides. Such sub-peptides are recognizable by assessing thesequence of the identified peptides for proteolytic cleavage sites of adesired protease. In one embodiment, tryptic peptides may includeadditional internal trypsin cleavage sites that can lead to sub-peptidesupon further cleavage by trypsin. In another embodiment, trypticpeptides may contain internal sites for proteases including, but notlimited to, trypsin GluC, AspN, chymotrypsin, and/or Lys C, which canlead to the formation of sub-peptides upon cleavage by any one, two, ormore of trypsin, GluC, AspN, chymotrypsin, and/or Lys C. In anotherembodiment, Lys C peptides may contain internal sites for otherproteases, such as GluC, AspN, chymotrypsin, and/or trypsin, which canlead to the formation of sub-peptides upon cleavage by any one, two, ormore of GluC, AspN, chymotrypsin, and/or trypsin. Such sub-peptides, andspecifically trypsin, GluC, AspN, chymotrypsin, and/or Lys C cleavagefragments of any one or more of the peptides set forth in SEQ ID Nos.1-39 are understood to be set forth and within the scope of thisdisclosure.

Embodiments set forth herein include compositions comprising one or moreof the peptides in Tables 1 and 2, and may optionally include peptidesthat are isotopically labeled but otherwise identical to one or more ofthe peptides found in Tables 1 and 2. In some embodiments, thecompositions comprise one or more, two or more, three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, or all thirty-nine (39) of the peptides in Tables 1 and 2. Suchcompositions may optionally include peptides, polypeptides, or proteinswhose amino acid sequence comprises peptides that are isotopicallylabeled but otherwise identical to one or more of the peptides found inTable 1 and Table 2. Where isotopically labeled synthetic or naturalpeptides, polypeptides, or proteins that comprise one, two, three, four,five, six or more of the peptides in Tables 1 and 2 are employed,protease treatment releases peptides that are isotopically labeled butotherwise identical to the peptides in Tables 1 and 2. Such isotopicallylabeled biological or biosynthetic peptides may be prepared, forexample, in programmed cell lysates or in tissue culture usingisotopically labeled amino acids. Each of the isotopically labeledpeptides may be labeled with one or more isotopes selected independentlyfrom the group consisting of: ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H orcombinations thereof. Compositions comprising peptides from the ALK,Ros, Ron, Ret, TS, and/or FGFR1 proteins, whether isotope labeled ornot, do not need to contain all of the peptides from that protein (e.g.,a complete set of tryptic peptides). In some embodiments thecompositions do not contain all peptides in combination from ALK, Ros,Ron, Ret, TS, and/or FGFR1 proteins, and particularly all of thepeptides appearing in Table 1 and Table 2. Compositions containingpeptides may be in the form of dried or lyophilized materials, liquid(e.g., aqueous) solutions or suspensions, arrays, or blots.

In one embodiment, the additional information about specific ALK, Ros,Ron, Ret, TS, and/or FGFR1 peptides includes one or more, two or more,or three or more of the mono isotopic mass of each peptide, itsprecursor charge state, the precursor m/z value, the m/z transitionions, and the ion type of each transition ion for peptides resultingfrom Lys C proteolysis of ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins.

In another embodiment, the additional information about specific ALK,Ros, Ron, Ret, TS, and/or FGFR1 peptides, includes one or more, two ormore, or three or more of the mono isotopic mass of each peptide, itsprecursor charge state, the precursor m/z value, the m/z transitionions, and the ion type of each transition ion for peptides resultingfrom trypsin proteolysis of ALK, Ros, Ron, Ret, TS, and/or FGFR1proteins.

In still another embodiment, the additional information about specificALK, Ros, Ron, Ret, TS, and/or FGFR1 peptides, includes one or more, twoor more, or three or more of the mono isotopic mass of each peptide, itsprecursor charge state, the precursor m/z value, the m/z transitionions, and the ion type of each transition ion for peptides resultingfrom trypsin and/or Lys C proteolysis of ALK, Ros, Ron, Ret, TS, and/orFGFR1 proteins. In one embodiment, a single tryptic and/or Lys Cproteolytic peptide from each of the ALK, Ros, Ron, Ret, TS, and/orFGFR1 proteins, along with the relevant additional information isemployed in a diagnostic determination. Thus, for example, the peptidesof SEQ ID NOs 4, 7, 9, 16, 31 and/or 36, and additional informationabout those peptides (see Table 3) in employed in a diagnostic analysis.

TABLE 3 Mono Precursor ALK Isotopic Charge Precursor Transition IonSEQ ID Peptide sequence Mass State m/z m/z Type SEQ ID NO: 4SNQEVLEFVTSGGR 1521.7423 2 761.878  723.378 y7 2 761.878  852.421 y8 2761.878  965.505 y9 2 761.878 1064.573 y10 Mono Precursor Ros IsotopicCharge Precursor Transition Ion SEQ ID Peptide sequence Mass State m/zm/z Type SEQ ID NO: 7 GEGLLPVR 839.4865 2 420.75 371.24 y3 2 420.75484.324 y4 2 420.75 654.429 y6 Mono Precursor Ron Isotopic ChargePrecursor Transition Ion SEQ ID Peptide sequence Mass State m/z m/z TypeSEQ ID NO: 9 ILQVELVR 968.6018 2 485.308 516.314 y4 2 485.308 615.382 y52 485.308 743.441 y6 2 485.308 856.525 y7 Mono Precursor Ret IsotopicCharge Precursor Transition Ion SEQ ID Peptide sequence Mass State m/zm/z Type SEQ ID NO: 16 ALSTWIENK 936.4552 2 579.811 390.198 y3 2 579.811503.282 y4 2 579.811 689.361 y5 2 579.811 790.409 y6 2 579.811 877.441y7 Mono Precursor TS Isotopic Charge Precursor Transition Ion SEQ IDPeptide sequence Mass State m/z m/z Type SEQ ID NO: 31 DEFPLLTTK1062.5597 2 532.287 462.292 y4 2 532.287 575.376 y5 2 532.287 672.429 y62 532.287 819.497 y7 Mono Precursor FGFR1 Isotopic Charge PrecursorTransition Ion SEQ ID Peptide sequence Mass State m/z m/z TypeSEQ ID NO: 36 DDVQSINWLR 1244.6149 2 623.315 588.325 y4 2 623.315701.409 y5 2 623.315 788.441 y6 2 623.315 916.499 y7

Certain Embodiments

Certain embodiments of the invention are described below.

-   1. A method for measuring the level of the ALK, Ros, Ron, Ret, TS,    and/or FGFR1 proteins in a biological sample, comprising detecting    and/or quantifying the amount of one or more modified and/or    unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment    peptides in a protein digest prepared from said biological sample    using mass spectrometry; and calculating the level of modified or    unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein in said    sample; and

wherein said amount is a relative amount or an absolute amount.

-   2. The method of embodiment 1, further comprising the step of    fractionating said protein digest prior to detecting and/or    quantifying the amount of one or more modified or unmodified ALK,    Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides.-   3. The method of embodiment 2, wherein said fractionating step is    selected from the group consisting of gel electrophoresis, liquid    chromatography, capillary electrophoresis, nano-reversed phase    liquid chromatography, high performance liquid chromatography, or    reverse phase high performance liquid chromatography.-   4. The method of any of embodiments 1-3, wherein said protein digest    of said biological sample is prepared by the Liquid Tissue™    protocol.-   5. The method of any of embodiments 1-3, wherein said protein digest    comprises a protease digest.-   6. The method of embodiment 5, wherein said protein digest comprises    a trypsin and/or lys C digest.-   7. The method of any of embodiments 1-6, wherein said mass    spectrometry comprises tandem mass spectrometry, ion trap mass    spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass    spectrometry, MALDI mass spectrometry, and/or time of flight mass    spectrometry.-   8. The method of embodiment 7, wherein the mode of mass spectrometry    used is Selected Reaction Monitoring (SRM), Multiple Reaction    Monitoring (MRM), and/or multiple Selected Reaction Monitoring    (mSRM), or any combination thereof.-   9. The method of any of embodiments 1 to 8, wherein the ALK, Ros,    Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises an    amino acid sequence as set forth as SEQ ID NO:1, SEQ ID NO:2, SEQ ID    NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID    NO:8 SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID    NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16. SEQ ID NO:17, SEQ    ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,    SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID    NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ    ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,    SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.-   10. The method of any of embodiments 1-9, wherein the biological    sample is a blood sample, a urine sample, a serum sample, an ascites    sample, a sputum sample, lymphatic fluid, a saliva sample, a cell,    or a solid tissue.-   11. The method of any of embodiments 1-10, wherein the biological    sample is formalin fixed tissue.-   12. The method of any of embodiments 1-11, wherein the biological    sample is paraffin embedded tissue.-   13. The method of any of embodiments 1-12, wherein the biological    sample is tissue that is obtained from a tumor.-   14. The method of embodiment 13, wherein the tumor is a primary    tumor.-   15. The method of embodiment 13, wherein the tumor is a secondary    tumor.-   16. The method of any of embodiments 1 to 15, further comprising    quantifying modified and/or unmodified ALK, Ros, Ron, Ret, TS,    and/or FGFR1 protein fragment peptides.-   17(a). The method of any of embodiments 1-16, wherein quantifying    the ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides    comprises comparing an amount of one or more ALK, Ros, Ron, Ret, TS,    and/or FGFR1 protein fragment peptides comprising an amino acid    sequence of about 8 to about 45 amino acid residues of ALK, Ros,    Ron, Ret, TS, and/or FGFR1 proteins in one biological sample to the    amount of the same ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein    fragment peptides in a different and separate sample or biological    sample.-   17(b). The method of any of embodiments 1-16, wherein quantifying    the ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides    comprises comparing an amount of one or more ALK, Ros, Ron, Ret, TS,    and/or FGFR1 protein fragment peptides comprising an amino acid    sequence of about 8 to about 45 amino acid residues of ALK, Ros,    Ron, Ret, TS, and/or FGFR1 proteins, as shown in SEQ ID NO:1, SEQ ID    NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID    NO:7, SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID    NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16. SEQ    ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,    SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID    NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ    ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,    SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, in one    biological sample to the amount of the same ALK, Ros, Ron, Ret, TS,    and/or FGFR1 protein fragment peptides in a different and separate    biological sample.-   18. The method of embodiment 17(a) or 17(b), wherein quantifying one    or more ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment    peptides comprises determining the amount of each of the ALK, Ros,    Ron, Ret, TS, and/or FGFR1 protein fragment peptides in a biological    sample by comparison to an added internal standard peptide of known    amount, wherein each of the ALK, Ros, Ron, Ret, TS, and/or FGFR1    protein fragment peptides in the biological sample is compared to an    added internal standard peptide having the same amino acid sequence.-   19. The method of embodiment 18, wherein the internal standard    peptide is an isotopically labeled peptide.-   20. The method of embodiment 19, wherein the isotopically labeled    internal standard peptide comprises one or more heavy stable    isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations    thereof.-   21. The method of any of embodiments 1-20, wherein detecting and/or    quantifying the amount of one or more modified or unmodified ALK,    Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in the    protein digest indicates the presence of modified and/or unmodified    ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein and an association with    cancer in a patient or subject.-   22. The method of embodiment 21, further comprising correlating the    results of said detecting and/or quantifying the amount of one or    more modified and/or unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1    protein fragment peptides, or the amount of said ALK, Ros, Ron, Ret,    TS, and/or FGFR1 proteins to the diagnostic stage/grade/status of    the cancer.-   23. The method of embodiment 22, wherein correlating the results of    said detecting and/or quantifying the amount of one or more modified    or unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment    peptides, or the amount of said ALK, Ros, Ron, Ret, TS, and/or FGFR1    proteins to the diagnostic stage/grade/status of the cancer is    combined with detecting and/or quantifying the amount of other    proteins or peptides from other proteins in a multiplex format to    provide additional information about the diagnostic    stage/grade/status of the cancer.-   24. The method of any one of embodiments 1-23, further comprising    selecting for a patient or subject from which said biological sample    was obtained a treatment based on the presence, absence, or amount    of one or more ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment    peptides or the amount of ALK, Ros, Ron, Ret, TS, and/or FGFR1    proteins.-   25. The method of any one of embodiments 1-24, further comprising    administering to a patient or subject from which said biological    sample was obtained a therapeutically effective amount of a    therapeutic agent, wherein the therapeutic agent and/or amount of    the therapeutic agent administered is based upon amount of one or    more modified or unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1    protein fragment peptides or the amount of ALK, Ros, Ron, Ret, TS,    and/or FGFR1 proteins.-   26. The method of embodiments 24 and 25, wherein the treatment or    the therapeutic agent is directed to cancer cells expressing ALK,    Ros, Ron, Ret, TS, and/or FGFR1 proteins.-   27. The method of embodiments 1-27, wherein the biological sample is    formalin fixed tumor tissue that has been processed for quantifying    the amount of one or more modified or unmodified ALK, Ros, Ron, Ret,    TS, and/or FGFR1 protein fragment peptides employing the Liquid    Tissue™ protocol and reagents.-   28. The method of any of embodiments 1-28, wherein said one or more    modified or unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein    fragment peptides is one or more of the peptides in Table 1.-   29. A composition comprising one or more, two or more, three or    more, four or more, five or more, six or more, seven or more, eight    or more, nine or more, or ten or more of the peptides in Table 1    and/or antibodies thereto.-   30. The composition of embodiment 30, comprising one or more, two or    more, three or more, four or more, five or more, six or more, seven    or more, eight or more, nine or more, or ten or more of the peptides    of Table 2 or antibodies thereto.

Exemplary SRM/MRM Assay Method

-   1. The method described below was used to: 1) identify candidate    peptides from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins that    can be used for a mass spectrometry-based SRM/MRM assay for the ALK,    Ros, Ron, Ret, TS, and/or FGFR1 proteins, 2) develop individual    SRM/MRM assay, or assays, for target peptides from the ALK, Ros,    Ron, Ret, TS, and/or FGFR1 proteins, and 3) apply quantitative    assays to cancer diagnosis and/or choice of optimal therapy.    Identification of SRM/MRM candidate fragment peptides for the ALK,    Ros, Ron, Ret, TS, and/or FGFR1 proteins:    -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins,        where individual fragment peptides do not contain any peptide        modifications such as phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins        that carry peptide modifications such as for example        phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length ALK, Ros, Ron, Ret, TS, and/or FGFR1        proteins potentially can be measured, but preferred peptides        used for development of the SRM/MRM assay are those that are        identified by mass spectrometry directly in a complex Liquid        Tissue™ protein lysate prepared from a formalin fixed biological        sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in a patient or subject tissue and which        ionize, and thus can be detected, in a mass spectrometer when        analyzing a Liquid Tissue™ lysate from a formalin fixed        biological sample are identified as candidate peptides for        assaying peptide modifications of the ALK, Ros, Ron, Ret, TS,        and/or FGFR1 proteins-   2. Mass Spectrometry Assay for Fragment Peptides from ALK, Ros, Ron,    Ret, TS, and/or FGFR1 proteins:    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue™        lysate is applied to peptides from the ALK, Ros, Ron, Ret, TS,        and/or FGFR1 proteins        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. An SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins        that is detected, as a function of the unique SRM/MRM signature        peak area from an SRM/MRM mass spectrometry analysis, can        indicate both the relative and absolute amount of the ALK, Ros,        Ron, Ret, TS, and/or FGFR1 proteins in a particular protein        lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by                comparing (a) the SRM/MRM signature peak area from a                given ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptide                detected in a Liquid Tissue™ lysate from one formalin                fixed biological sample to (b) the same SRM/MRM                signature peak area of the same ALK, Ros, Ron, Ret, TS,                and/or FGFR1 fragment peptide in at least a second,                third, fourth or more Liquid Tissue™ lysates from at                least a second, third, fourth or more formalin fixed                biological samples            -   2. Determining increased or decreased presence of the                ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by                comparing (a) the SRM/MRM signature peak area from a                given ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptide                detected in a Liquid Tissue™ lysate from one formalin                fixed biological sample to (b) SRM/MRM signature peak                areas developed from fragment peptides from other                proteins, in other samples derived from different and                separate biological sources, where the SRM/MRM signature                peak area comparison between the 2 samples for a peptide                fragment is normalized to amount of protein analyzed in                each sample.            -   3. Determining increased or decreased presence of the                ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by (a)                comparing the SRM/MRM signature peak area for a given                ALK, Ros, Ron, Ret, TS, and/or FGFR1 peptide to (b) the                SRM/MRM signature peak areas from other fragment                peptides derived from different proteins within the same                Liquid Tissue™ lysate from the formalin fixed biological                sample in order to normalize changing levels of ALK,                Ros, Ron, Ret, TS, and/or FGFR1 proteins to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and to modified fragment peptides of                the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins, where                the modifications include but are not limited to                phosphorylation and/or glycosylation, and where the                relative levels of modified peptides are determined in                the same manner as determining relative amounts of                unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing (a) the SRM/MRM signature peak area for a given            fragment peptide from the ALK, Ros, Ron, Ret, TS, and/or            FGFR1 proteins in an individual biological sample to (b) the            SRM/MRM signature peak area of an internal fragment peptide            standard spiked into the protein lysate from the biological            sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the ALK, Ros, Ron, Ret, TS,                and/or FGFR1 proteins that is being interrogated. This                standard is spiked into a sample in known amounts, and                the SRM/MRM signature peak area can be determined for                both the internal fragment peptide standard and the                native fragment peptide in the biological sample                separately, followed by comparison of both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.-   3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and    Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the ALK, Ros, Ron, Ret, TS, and/or FGFR1        proteins and demonstrate that the previously-determined        association, as well understood in the field of cancer, of ALK,        Ros, Ron, Ret, TS, and/or FGFR1 protein expression to the        stage/grade/status of cancer in patient or subject tumor tissue        is confirmed    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the ALK, Ros, Ron, Ret, TS, and/or FGFR1        proteins and demonstrate correlation with clinical outcomes from        different treatment strategies, wherein this correlation has        already been demonstrated in the field or can be demonstrated in        the future through correlation studies across cohorts of        patients or subjects and tissue from those patients or subjects.        Once either previously established correlations or correlations        derived in the future are confirmed by this assay then the assay        method can be used to determine optimal treatment strategy        A Mass Spectrometry Assay for Fragment Peptides from ALK, Ros,        Ron, Ret, TS, and/or FGFR1 Proteins    -   a. SRM/MRM assay to determine the amount of the fragment peptide        of the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins that is        detected to determine the relative and/or absolute amount of the        ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins in a protein        lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by                comparing (a) the SRM/MRM signature peak area from a                given ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein                peptide detected in a Liquid Tissue™ lysate from one                formalin fixed biological sample to (b) the same SRM/MRM                signature peak area of the same ALK, Ros, Ron, Ret, TS,                and/or FGFR1 protein fragment peptide in at least a                second, third, fourth or more Liquid Tissue™ lysates                from at least a second, third, fourth or more formalin                fixed biological samples            -   2. Determining increased or decreased presence of the                ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by                comparing (a) the SRM/MRM signature peak area from a                given ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein                peptide detected in a Liquid Tissue™ lysate from one                formalin fixed biological sample to (b) SRM/MRM                signature peak areas developed from fragment peptides                from other proteins, in other samples derived from                different and separate biological sources, where the                SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment is normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins by                comparing (a) the SRM/MRM signature peak area for a                given ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein                peptide to (b) the SRM/MRM signature peak areas from                other fragment peptides derived from different proteins                within the same Liquid Tissue™ lysate from the formalin                fixed biological sample in order to normalize changing                levels of ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins                to levels of other proteins that do not change their                levels of expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins, where                the modifications include but are not limited to                phosphorylation and/or glycosylation, and where the                relative levels of modified peptides are determined in                the same manner as determining relative amounts of                unmodified peptides.        -   ii. Absolute quantitation of a given peptide or the protein            from which it is derived, may be achieved by comparing (a)            the SRM/MRM signature peak area for a given fragment peptide            from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins in an            individual biological sample to (b) the SRM/MRM signature            peak area of an internal fragment peptide standard spiked            into the protein lysate from the biological sample.

The internal standard can be a labeled synthetic version of the fragmentpeptide from the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins that isbeing interrogated (or a protein or polypeptide comprising the labeledsynthetic version of the fragment peptide that is released uponproteolysis). The standard is spiked into a sample in known amounts, andthe SRM/MRM signature peak area can be determined for both the internalfragment peptide standard and the native fragment peptide in thebiological sample separately, followed by comparison of both peak areas.

This can be applied to unmodified fragment peptides and modifiedfragment peptides, where the modifications include but are not limitedto phosphorylation and/or glycosylation, and where the absolute levelsof modified peptides can be determined in the same manner as determiningabsolute levels of unmodified peptides.

Assessment of ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein levels intissues based on analysis of formalin fixed patient-derived orsubject-derived tissue can provide diagnostic, prognostic, andtherapeutically-relevant information about each particular patient orsubject. Described herein is a method for measuring the levels of theALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins in a biological sample,comprising detecting and/or quantifying the amount of one or moremodified or unmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteinfragment peptides in a protein digest prepared from said biologicalsample using mass spectrometry; and calculating the level of modified orunmodified ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins in said sample;and wherein said level is a relative level or an absolute level. In arelated embodiment, quantifying one or more ALK, Ros, Ron, Ret, TS,and/or FGFR1 protein fragment peptides comprises determining the amountof each of the ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragmentpeptides in a biological sample by comparison to an added internalstandard peptide of known amount, wherein each of the ALK, Ros, Ron,Ret, TS, and/or FGFR1 protein fragment peptides in the biological sampleis compared to an internal standard peptide having the same amino acidsequence. In some embodiments the internal standard is an isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or a combinationthereof.

The method for measuring levels of the ALK, Ros, Ron, Ret, TS, and/orFGFR1 proteins in a biological sample described herein (or fragmentpeptides as surrogates thereof) may be used as a diagnostic indicator ofcancer in a patient or subject. In one embodiment, the results frommeasurements of levels of the ALK, Ros, Ron, Ret, TS, and/or FGFR1proteins may be employed to determine the diagnostic stage/grade/statusof a cancer by correlating (e.g., comparing) the levels of ALK, Ros,Ron, Ret, TS, and/or FGFR1 proteins found in a tissue with the levels ofALK, Ros, Ron, Ret, TS, and/or FGFR1 proteins found in normal and/orcancerous or precancerous tissues.

The only current method in use for detecting levels of specific proteinsin formalin fixed patient tissue is immunohistochemistry (IHC). Thismethod analyzes only one protein at a time on a single tissue sectionfrom a patient tumor tissue sample. Thus, in order to analyze multipleproteins, multiple tissue sections must be interrogated, which costsmuch time and labor. IHC uses an antibody to detect the presence of thetarget protein and, because of the potential for non-specific binding ofthe antibody to proteins there is great inherent potential for signalbackground in any IHC experiment. In addition, IHC is onlysemi-quantitative at best. Due to these problems IHC fails to providefor objective quantitative analysis of multiple proteins simultaneously.The methods described here are able to provide for objectivequantitation of the ALK, Ros, Ron, Ret, TS, and/or FGFR1 proteinssimultaneously with 100% assay specificity utilizing a single section ofa patient tissue sample, saving significant time and money whileproviding for much more valuable data about expression of the ALK, Ros,Ron, Ret, TS, and/or FGFR1 proteins.

This multiplex SRM/MRM assay can also include simultaneous analysis ofother additional proteins beyond the ALK, Ros, Ron, Ret, TS, and/orFGFR1 proteins, including drug target proteins such as EGFR, IGF-1R, andcMet. This is valuable because analysis of additional proteins alsoindicates which additional drugs might be useful for treating aparticular cancer. Examples of additional drugs based on analysis ofadditional example drug target proteins include Erbitux, which targetsthe EGFR receptor, Figitumumab, which targets IGF-1R, and Foretinib,which targets c-Met and vascular endothelial growth factor receptor 2(VEGFR-2).

Because both nucleic acids and protein can be analyzed from the sameLiquid Tissue™ biomolecular preparation, it is possible to generateadditional information about disease diagnosis and drug treatmentdecisions from the nucleic acids in the same sample used for proteinanalysis. For example, if the ALK, Ros, Ron, Ret, TS, and/or FGFR1proteins are expressed by certain cells at increased levels, whenassayed by SRM the data can provide information about the state of thecells and their potential for uncontrolled growth, potential drugresistance and the development of cancers can be obtained. At the sametime, information about the status of the ALK, Ros, Ron, Ret, TS, and/orFGFR1 genes and/or the nucleic acids and proteins they encode (e.g.,mRNA molecules and their expression levels or splice variations) can beobtained from nucleic acids present in the same biomolecularpreparation. In one embodiment, information about the ALK, Ros, Ron,Ret, TS, and/or FGFR1 proteins and/or one, two, three, four or moreadditional proteins may be assessed by examining the nucleic acidsencoding those proteins. Those nucleic acids can be examined, forexample, by one or more, two or more, or three or more of: sequencingmethods, polymerase chain reaction methods, restriction fragmentpolymorphism analysis, identification of deletions, insertions, and/ordeterminations of the presence of mutations, including but not limitedto, single base pair polymorphisms, transitions, transversions, orcombinations thereof.

The above description and exemplary embodiments of methods andcompositions are illustrative of the scope of the present disclosure.Because of variations which will be apparent to those skilled in theart, however, the present disclosure is not intended to be limited tothe particular embodiments described above.

1. A method for measuring the level of at least one of the human Ros, Ron, Ret, TS, and/or FGFR1 proteins in a human biological sample of formalin-fixed tissue, comprising detecting and quantifying using mass spectrometry the amount of Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptide in a protein digest prepared from said human biological sample; and calculating the level of Ros, Ron, Ret, TS, and/or FGFR1 protein in said sample; wherein said amount is a relative amount or an absolute amount.
 2. The method of claim 1, further comprising the step of fractionating said protein digest prior to detecting and quantifying the amount of said Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides.
 3. (canceled)
 4. The method of claim 1, wherein said protein digest comprises a protease digest.
 5. The method of claim 1, wherein said mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry.
 6. The method of claim 1, wherein the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises an amino acid sequence as set forth as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16. SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. 7-8. (canceled)
 9. The method of claim 1 wherein quantifying the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises comparing an amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in one biological sample to the amount of the same ALK, Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in a different and separate biological sample.
 10. The method of claim 1, wherein quantifying one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides comprises determining the amount of each of the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence.
 11. The method of claim 10, wherein the internal standard peptide is an isotopically labeled peptide.
 12. The method of claim 1, wherein detecting and quantifying the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides in the protein digest indicates the presence of Ros, Ron, Ret, TS, and/or FGFR1 protein and an association with cancer, including lung cancer, in a patient or subject.
 13. The method of claim 12, further comprising correlating the results of said detecting and quantifying the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides, or the amount of said Ros, Ron, Ret, TS, and/or FGFR1 proteins to the diagnostic stage/grade/status of the cancer, including lung cancer.
 14. The method of claim 13, wherein correlating the results of said detecting and quantifying the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides, or the amount of said Ros, Ron, Ret, TS, and/or FGFR1 proteins to the diagnostic stage/grade/status of the cancer is combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer, including lung cancer.
 15. The method of claim 13, further comprising administering to a patient or subject from which said biological sample was obtained a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon the amount of one or more Ros, Ron, Ret, TS, and/or FGFR1 protein fragment peptides or the amount of Ros, Ron, Ret, TS, and/or FGFR1 proteins.
 16. The method of claim 15, wherein the treatment or the therapeutic agent is directed to cancer cells expressing Ros, Ron, Ret, TS, and/or FGFR1 proteins.
 17. (canceled) 